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Article

Genome-Wide Identification and Characterization of AGO, DCL, and RDR Gene Families in Siraitia grosvenorii

by
Yimei Zang
1,
Chongnan Wang
1,
Jiaxian Su
1,
Changming Mo
2,3,
Lei Xie
1,
Zuliang Luo
1,* and
Xiaojun Ma
1,*
1
State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
2
Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
3
Yuelushan Laboratory, Changsha 410006, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(11), 5301; https://doi.org/10.3390/ijms26115301
Submission received: 16 April 2025 / Revised: 25 May 2025 / Accepted: 29 May 2025 / Published: 30 May 2025
(This article belongs to the Section Molecular Plant Sciences)
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Versions Notes

Abstract

RNA silencing regulates diverse cellular processes in plants. Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) proteins are core components of RNA interference (RNAi). Despite their functional significance, the systematic identification and characterization of these families have remained largely unexplored in Siraitia grosvenorii. Using HMMER and Pfam analyses, we identified six SgAGO, four SgDCL, and six SgRDR genes. Phylogenetic analysis classified SgAGOs, SgDCLs, and SgRDRs into five, four, and four clades, respectively, all of which clustered closely with homologs from other Cucurbitaceae species, demonstrating lineage-specific evolutionary conservation. Promoter cis-element analysis revealed the significant enrichment of hormonal (methyl jasmonate, abscisic acid) and stress-responsive (light, hypoxia) elements, indicating their roles in environmental adaptation. Tissue-specific expression profiling showed that most SgAGO, SgDCL, and SgRDR genes were highly expressed in flowers and mid-stage fruits (35 days after pollination), while SgAGO10.1 exhibited stem-specific expression. By contrast, SgRDR1.2 displayed no tissue specificity. Notably, sex-biased expression patterns in dioecious flowers suggested the RNAi-mediated regulation of gametophyte development and their potential roles in reproductive and secondary metabolic processes. This study lays the foundation for further exploration of RNAi machinery’s role in coordinating mogroside biosynthesis and stress resilience in S. grosvenorii while providing potential targets for genetic improvement.

1. Introduction

RNA silencing is a highly conserved regulatory mechanism in eukaryotes involving small non-coding RNAs that regulate gene expression at both transcriptional and post-transcriptional levels [1,2]. This mechanism plays a crucial role in modulating plant growth, development, antiviral defense, and stress responses [3]. The core components of the RNA interference (RNAi) machinery include the Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) protein families. These proteins collaborate to process double-stranded RNAs (dsRNAs) into small interfering RNAs (siRNAs) or microRNAs (miRNAs), which subsequently guide the RNA-induced silencing complex (RISC) to target mRNAs for cleavage or translational inhibition [2].
The AGO protein family serves as the central effector of the RISC, mediating gene silencing through guide strand binding of small RNAs (sRNAs), such as siRNAs, which leads to targeted mRNA cleavage or translational repression [4,5]. Structurally, AGO proteins are characterized by conserved functional domains, including Argo-N/Argo-L, DUF 1785, PAZ, ArgoMid, and PIWI [6]. Among these, the PAZ domain contains a critical RNA-binding site [7], while the ArgoMid domain interacts with the 5′-terminal phosphodiester bond of sRNAs [8]. The PIWI domain, which confers endonuclease activity, binds the 5′ end of siRNAs, thereby enabling RNA-guided target recognition and silencing [5].
DCL proteins, on the other hand, are ribonuclease III enzymes responsible for cleaving dsRNAs into 21–24 nucleotide sRNAs [9]. There are typically four DCL proteins in plants, each with specific roles in producing different types of sRNAs [10]. DCL proteins possess six conserved and functional domains: DEAD, Helicase-C, DUF1785, PAZ, RNase III, and DSRM. These domains are essential for the proteins to exert their functions [11,12].
RDR proteins, a major RNAi-related protein group, synthesize dsRNA using single-stranded RNA as a template. This dsRNA serves as a substrate for DCL enzymes to generate secondary siRNAs [13,14]. RDR proteins are characterized by a conserved RNA-dependent RNA polymerase (RdRP) domain [15,16,17], which is integral to their function as members of the RNA interference gene family [18].
Genome-wide studies of the AGO, DCL, and RDR families across plant species have elucidated their evolutionary conservation and functional divergence. In model plants, these families exhibit lineage-specific expansion: Arabidopsis (Arabidopsis thaliana) harbors 10 AGOs, 4 DCLs, and 6 RDRs [19], while rice (Oryza sativa) [20] and maize (Zea mays) [21] possess 18 AGOs, 4 DCLs, and 6 RDRs and 18 AGOs, 5 DCLs, and 11 RDRs, respectively. Members within each family display distinct spatiotemporal expression patterns and regulate diverse biological processes. Advances in genomics and computational tools have further enabled systematic analyses in non-model species, including fruit crops such as sweet orange (Citrus sinensis) [22], strawberry (Fragaria spp.) [23], banana (Musa acuminata) [7], grapevine (Vitis vinifera) [24], peach (Prunus persica L.) [25], pepper (Capsicum annuum L.) [16], and cucumber (Cucumis sativus L.) [26]. These investigations have delineated evolutionary trajectories, structural variations, and expression dynamics of RNAi-related genes, offering critical insights into the adaptation and diversification of RNAi machinery in horticulturally significant species.
Siraitia grosvenorii, commonly known as luohanguo or monk fruit, is an edible horticultural plant belonging to the family Cucurbitaceae. It is renowned for its high content of mogrosides, which are natural sweeteners with zero calories and a sweetness hundreds of times more intense than sucrose [27]. Despite its economic and medicinal importance, the AGO, DCL, and RDR gene families in S. grosvenorii remain largely uncharacterized. Identifying and characterizing these gene families in S. grosvenorii will provide valuable insights into RNAi machinery and its role in regulating gene expression, growth, development, and the accumulation of mogrosides in this species.
In this study, we performed a genome-wide identification and expression analysis of the SgAGO, SgDCL, and SgRDR gene families in S. grosvenorii. We employed bioinformatic approaches to search for candidate genes in the S. grosvenorii genome and analyzed their phylogenetic relationships, conserved domains, gene structures, and chromosomal locations. Furthermore, we investigated the transcript abundances of SgAGO, SgDCL, and SgRDR genes in the roots, stems, leaves, flowers, and fruits at various time points after pollination (DAP) using quantitative real-time PCR (qRT-PCR). This study provides insights into the RNAi machinery in S. grosvenorii and paves the way for future functional studies of these gene families in this economically and medicinally important plant species.

2. Results

2.1. Identification and Structural Analysis of SgAGO, SgDCL, and SgRDR Genes in S. grosvenorii

Using a HMMER search, we identified the putative SgAGO, SgDCL, and SgRDR genes of S. grosvenorii. The resulting sequences were further confirmed by analyzing conserved domains as putative family members according to the Pfam database [28]. As a result, we identified a total of six AGO genes, four DCL genes, and six RDR genes in S. grosvenorii. The detailed characteristics of all genes identified in this study, including the number of amino acids, molecular weight (kDa), theoretical pI, subcellular location, open reading frame (ORF) length, protein length (amino acid, aa), and grand average of hydropathicity (GRAVY), are listed in Table 1. The polypeptide lengths of the six identified SgAGO genes ranged from 927 to 1056 amino acids, with predicted molecular weights ranging from 105.33 to 117.11 kDa and theoretical pI values from 8.79 to 9.48. Four SgDCL genes encoded polypeptides that ranged from 1628 to 1952 amino acids in length, with molecular weights between 183.60 and 219.66 kDa, and theoretical pI values ranging from 5.84 to 8.55. Moreover, six SgRDR genes encoded polypeptides that ranged from 1026 to 1133 amino acids in length, with molecular weights between 116.35 and 135.98 kDa, and theoretical pI values ranging from 6.72 to 8.30.
Conserved domain analysis via the Pfam database revealed that all SgAGO proteins shared an N-terminus PAZ domain and a C-terminus PIWI super family domain, which are the core properties of plant AGO proteins. Moreover, conserved domain analysis also showed that the ArgoL1, Argo-N, and DUF1785 domains were present in all identified SgAGO proteins. Furthermore, the Gly-rich Ago1 domain was found in SgAGO1, which is conserved in AtAGO1. Interestingly, we observed that the Argo Mid and Argo-L2 domains were present in all SgAGO variants, except SgAGO7 (Figure 1A(I,III)).
All SgDCL proteins possessed four types of conserved domains, such as Helicase_C, Helicase Superfamily 1/2, Dicer_dimer, PAZ, RNase III, and P-loop. Furthermore, SgDCL2, SgDCL3, and SgDCL 4 contained DEAD/DEAH, and a double-stranded RNA-binding domain (DSRM) was present in SgDCL1, SgDCL 2, and SgDCL 4, similar to AtDCLs. In addition, SgDCL2 contained Tic20 (Figure 1B(I,III)).
The six newly identified SgRDR proteins shared a common domain consisting of a sequence motif that corresponded to the catalytic β’ subunit of RdRP [29]. In addition, SgRDR1.2, SgRDR1.3, SgRDR2, and SgRDR6 shared another common sequence motif, the RRM domain (Figure 1C(I,III)).

2.2. Motif and Domain Analyses of SgAGO, SgDCL, and SgRDR in S. grosvenorii

Conserved motifs within the SgAGO, SgDCL, and SgRDR protein sequences of S. grosvenorii and A. thaliana were identified using the Multiple Em for Motif Elicitation (MEME) motif discovery tool. Comparative analysis revealed that 10 out of 20 conserved motifs were shared between AGO proteins from A. thaliana and S. grosvenorii (Figure 1A(II)). Notably, motif 15 was absent in SgAGO4, a pattern consistent with its Arabidopsis homologs AtAGO4 and AtAGO8. Furthermore, motif 3 was exclusively present in SgAGO1/5/6/10, which is also conserved in their Arabidopsis counterparts (AtAGO1/5/6/10), suggesting that motif 3 may represent a signature motif specific to the AGO1/5/6/10 subclade.
Within the DCL protein family, 13 out of 15 conserved motifs were universally identified across all DCL members (Figure 1B(II)). Notably, SgDCL2 lacked motif 15, a structural feature shared with its Arabidopsis homolog AtDCL2. Furthermore, SgDCL3 uniquely lacked motif 14 compared to other SgDCLs.
Within the RDR protein family, MEME analysis identified seven conserved motifs, including motifs 8, 12, 6, 9, 3, 2, 1, and 5, as major structural features shared among RDR members (Figure 1C(II)). Notably, compared to other SgRDRs, SgRDR5 lacked seven conserved motifs and harbored an additional motif, motif 13, near the N-terminus. Furthermore, motif 15 was positioned at the C-terminus of SgRDRs, a structural pattern shared with its Arabidopsis homologs AtRDR3/4/5.

2.3. Phylogenetic Analysis of SgAGO, SgDCL, and SgRDR in S. grosvenorii and Other Plant Species

To elucidate the evolutionary relationships of SgAGO proteins, a phylogenetic tree was constructed using 78 protein sequences, which resolved into four major clades, AGO2/3/7, AGO4/6/8/9/15/16, AGO5/11/12/13/14, and AGO1/10, with an additional lineage-specific clade designated as AGO18 (Figure 2A). Each clade further diverged into monocot- and dicot-specific subclades. Notably, AGO18 formed a monocot-exclusive clade, suggesting functional divergence unique to monocots. Similarly, AGO11/12/13/14 clustered within a monocot-specific subclade yet shared ancestral affinity with the dicot AGO5 clade, indicating their origin from a common progenitor prior to monocot–dicot divergence. For the DCL family, phylogenetic analysis of 35 protein sequences delineated into four clades: DCL1, DCL2, DCL3, and DCL4 (Figure 2B). Each clade bifurcated into monocot and dicot lineages. The RDR phylogeny, comprising 49 protein sequences, resolved into four clades: RDR1, RDR2, RDR3/4/5, and RDR6 (Figure 2C). Intriguingly, ZmRDR3/4/5 and SHL2 clustered within the dicot RDR6 clade, implying a closer evolutionary relationship between these lineages.
Phylogenetic analysis revealed that SgAGO proteins clustered into four dicot-specific subclades, SgDCLs spanned all four canonical clades, and SgRDRs were assigned to four distinct clades, all demonstrating strong evolutionary conservation and close phylogenetic relationships with their homologs from other Cucurbitaceae species; notably, the majority of these genes formed a monophyletic branch with M. charantia, indicating close evolutionary relationships between S. grosvenorii and M. charantia in the SgAGO, SgDCL, and SgRDR families.

2.4. Protein–Protein Interactions of SgAGO, SgDCL, and SgRDR

A protein–protein interaction network for SgAGO, SgDCL, and SgRDR was constructed using the STRING 12 tool based on homologous proteins from A. thaliana. String mapping revealed that SgAGO, SgDCL, and SgRDR proteins aligned closely with their respective A. thaliana orthologs (Table 2).
Protein–protein interaction (PPI) network analysis was performed with an interaction score threshold of 0.7, and k-means clustering was employed to partition the proteins into three distinct clusters (Figure 3). Among the 16 analyzed proteins, Cluster 1 comprised SgAGO1/5/7/10, SgDCL1/4, and SgRDR1; Cluster 2 included SgAGO4, SgDCL2/3, and SgRDR2; and Cluster 3 exclusively contained SgRDR5. Strong intra-cluster interactions were observed in Clusters 1 and 2, suggesting that combinatorial associations of SgDCLs, SgAGOs, and SgRDRs may orchestrate distinct RNA silencing pathways in S. grosvenorii. Notably, SgRDR5 formed a solitary cluster (Cluster 3) and exhibited minimal interaction partners, implying its specialized functional role or regulatory divergence.

2.5. Cis-Acting Element Analysis in SgAGO, SgDCL, and SgRDR Genes

Cis-elements involved in hormone response, light response, stress response, and tissue specificity were identified in the 2000 bp upstream regulatory regions of the SgAGO, SgDCL, and SgRDR genes using the PlantCARE database (Figure 4, Table S5).
A total of 101 hormone-responsive elements (HREs) were identified across 375 promoter sequences of SgDCLs, SgRDRs, and SgDCLs in S. grosvenorii. Among these, 36 abscisic acid (ABA)-responsive elements were distributed as follows: 18 in SgAGOs, 3 in SgDCLs, and 15 in SgRDRs. Six auxin-responsive elements were detected, with two localized in SgAGOs, two in SgDCLs, and two in SgRDRs. For gibberellin (GA)-responsive elements, ten were identified, including four in SgAGOs, four in SgDCLs, and two in SgRDRs. Notably, 42 methyl jasmonate (MeJA)-responsive elements exhibited the highest abundance, with 16 in SgAGOs, 8 in SgDCLs, and 18 in SgRDRs. By contrast, only seven salicylic acid (SA)-responsive elements were observed, distributed as two in SgAGOs, one in SgDCLs, and four in SgRDRs. This distinct enrichment pattern suggested potential hormonal regulation biases in the RNAi machinery, particularly toward MeJA and ABA signaling pathways.
Additionally, a substantial number of stress-related elements (244/375) were observed, including those responsive to anaerobic or anoxic inducibility (34/244), drought (7/244), low temperature (7/244), light (186/244), defense (8/244), and wounding (2/44). Furthermore, tissue development-related and bioanabolic-responsive elements (26/375) were identified, such as those involved in endosperm expression (3/26), meristem expression (11/26), zein metabolism regulation (7/26), flavonoid biosynthesis regulation (1/26), and circadian control (4/26). Notably, the MYBHv1-binding site (4/375) was only observed on SgDCL2/3/4 promoter sequences. These results indicated that the SgAGO, SgDCL, and SgRDR genes may play significant roles in responding to hormones and light stress in S. grosvenorii.

2.6. Tissue-Specific Expression Patterns of SgAGO, SgDCL, and SgRDR Genes

To explore their potential roles in plant growth and development, we analyzed the expression patterns of SgAGO, SgDCL, and SgRDR genes across roots, stems, leaves, female flowers, and male flowers at three fruit ripening stages of S. grosvenorii. The spatial expression data, normalized with S. grosvenorii ubiquitin (SgUBQ), were compared to the root tissue data.
Relative to root expression, SgAGO gene family members generally exhibited low expression in roots, leaves, and 5 DAP fruits, while showing higher expression levels in stems, female flowers, male flowers, and mid-to-late-stage fruits. Notably, SgAGO1/5/7 displayed female flower-specific expression, with their transcript levels markedly surpassing those in other tissues. SgAGO6 showed dual-tissue preference, peaking in male flowers followed by female flowers, both significantly higher than in other organs. SgAGO10.1 was specifically upregulated in stems. During fruit development, SgAGO4 reached peak expression at 35 DAP, while SgAGO10.2 showed a unique temporal regulation pattern with maximal expression at 65 DAP. SgDCL gene family members were broadly repressed in leaves and 5 DAP fruits but strongly activated in female flowers and mid-to-late-stage fruits relative to their expression in root tissues. Tissue-specific divergence was observed: SgDCL1/4 were predominantly expressed in female flowers, whereas SgDCL2/3 peaked in 35 DAP fruits. Intriguingly, all SgDCLs maintained stable expression levels in stems. SgRDR gene family members were weakly expressed in roots and leaves but highly enriched in female flowers and mid-to-late-stage fruits as compared to roots. SgRDR2/6 exhibited striking female flower specificity. SgRDR1.1/1.3/5 were predominantly expressed in 35 DAP fruits. By contrast, SgRDR1.2 displayed broad activity with minimal tissue specificity (Figure 5).

3. Discussion

S. grosvenorii (Luo Han Guo), a perennial Cucurbitaceae vine, is renowned for its abundance of nutritionally and pharmacologically valuable compounds, including fatty acids, essential amino acids, flavonoids, and triterpenoids. Of particular interest are mogrosides, a group of intensely sweet triterpenoid secondary metabolites with non-caloric properties which hold promising applications in functional foods, natural sweeteners, and traditional medicine [30]. Despite its commercial potential, S. grosvenorii cultivation remains restricted to narrow geographic regions, with mogroside biosynthesis, plant growth, and yield being highly sensitive to environmental fluctuations [31]. Therefore, identifying these core regulatory genes and investigating their expression patterns across different tissues and developmental stages becomes imperative. Studies in A. thaliana have established that the core machinery of RNAi pathways involves three conserved gene families: AGO, DCL, and RDR [32]. In this study, we performed genome-wide identification and functional characterization of SgAGO, SgDCL, and SgRDR genes in S. grosvenorii.

3.1. Evolution and Functional Divergence Mechanisms of RNAi Pathway Gene Families

In this study, through domain and phylogenetic analyses of the SgAGO, SgDCL, and SgRDR gene families in S. grosvenorii, we have preliminarily revealed the expansion patterns and conserved mechanisms of functional divergence of these gene families during evolution. Whole genome duplication (WGD) and tandem duplication are the main mechanisms driving gene family expansion. In the SgAGO family, the formation of multiple subfamilies, such as AGO2/3/7 and AGO4/6/8/9/15/16 (Figure 2A), and the monophyletic clustering of homologous genes with M. charantia, suggest that the ancestor of Cucurbitaceae may have undergone an ancient polyploid event. This WGD provides raw material for gene family expansion, allowing genes to explore new functions without immediately affecting organism survival [33]. Additionally, the close phylogenetic relationship between RDR1.2 and RDR1.3 in the SgRDR family (Figure 2C) and their shared RRM domain (Figure 1C(I)) indicate that tandem duplication in local chromosomal regions may have driven the expansion of this subfamily. These local duplication events often lead to gene clustering on chromosomes, promoting functional specialization within the subfamily.
After gene family expansion, domain changes become an important driving force for functional divergence. In the SgAGO family, members typically contain conserved domains such as the N-terminal domain, PAZ, and Piwi. However, SgAGO7 specifically lacks the Mid and Argo-L2 domains (Figure 1A(I)), which may result in the loss of its typical RNA-binding function. By contrast, SgAGO1–6, retaining the PAZ and PIWI domains, are likely to perform core RNAi functions. This domain difference reflects the occurrence of subfunctionalization, where different members retain partial functions of the ancestral gene during evolution, thereby achieving functional specialization. Moreover, neofunctionalization events have also emerged within the gene families. For instance, the Tic20 domain unique to SgDCL2 (Figure 1B(I)) has not been reported in other DCL homologs. This may endow SgDCL2 with unique substrate recognition capabilities, such as involvement in chloroplast RNA processing. The emergence of this new function may provide a novel selective advantage for plants to adapt to specific environmental conditions [34].
From a phylogenetic perspective, the SgAGO, SgDCL, and SgRDR families exhibit both evolutionary conservation and species-specific adaptations. SgAGO18 forms a monocot-specific clade (Figure 2A), suggesting it may have undergone unique functional divergence to meet the special physiological demands of monocots. The differentiation of SgDCL and SgRDR families between monocots and dicots (Figure 2B,C) reveals that RNAi pathway genes have balanced functional conservation with lineage-specific adaptations during plant evolution. Notably, the close clustering of SgAGO, SgDCL, and SgRDR genes with their counterparts in M. charantia (Figure 2) suggests that these RNAi genes may have undergone coordinated functional evolution within the family Cucurbitaceae [34]. This coordinated evolution may be related to their shared regulatory demands for secondary metabolism, potentially enhancing the adaptability of Cucurbitaceae plants.
Further domain analysis provided crucial insights into the functional diversification of SgAGO, SgDCL, and SgRDR proteins. In the SgAGO family, in addition to the aforementioned specific domain loss in SgAGO7, SgAGO1 was identified to have a glycine-rich Argo domain, a signature characteristic of AGO1 [32,35]. For DCL family proteins, all SgDCL members possess six conserved domains: Helicase_C, Helicase Superfamily 1/2, Dicer_dimer, PAZ, RNase III, and P-loop. Interestingly, SgDCL1 lacks the DEAD/DEAH domain, similar to AtDCL1 in A. thaliana and CsDCL1b in C. sinensis [35]. By contrast, SgDCL3 lacks the DSRM domain, resembling AtDCL3 in A. thaliana and CaDCL3 in C. annuum [16]. Significantly, SgDCL4 retains the PAZ domain, consistent with AtDCL4 in A. thaliana [36], but markedly different from CsDCL4 in C. sinensis [35] and HaDCL4 in sunflower (Helianthus annuus) [17]. Regarding the RDR protein family, all six SgRDR members harbor the conserved RdRP domain. Additionally, an RRM is present in the upstream regions of SgRDR1.2, SgRDR1.3, SgRDR2, and SgRDR6, in line with previous reports on CsRDR2 in C. sinensis [35] and SmRDR2 in Salvia miltiorrhiza [37]. These conserved and specific changes in domains collectively underpin the molecular basis for gene family functional diversification and evolution. This will enhance our comprehensive understanding of the evolution and functional mechanisms of plant RNAi pathway gene families.

3.2. Cis-Acting Element Analysis Reveals Hormonal and Stress Response Regulatory Mechanisms of RNAi Core Genes in S.grosvenorii

This study systematically identified cis-acting elements in the promoter regions of SgAGO, SgDCL, and SgRDR genes in S. grosvenorii through bioinformatics analysis and preliminarily explored their potential functions in hormonal responses and stress adaptation. The results show that the promoter regions of these three gene families contain a total of 375 cis-acting elements, with hormone-responsive elements accounting for 27.2% (101/375), including regulatory elements for key phytohormones such as ABA, auxin, GA, MeJA, and SA. Notably, ABA and MeJA response elements were prominently distributed in SgAGOs and SgRDRs: the SgAGO family was enriched with 50% ABA elements (18/36), while the SgRDR family carried 42.9% MeJA elements (18/42). Previous studies have revealed that OsRDR6 in rice is positively regulated by ABA and mediates ABA-induced siRNA-dependent amplification and silencing of isocitrate lyase (ICL) transcripts [38]. Similarly, FaRDR1k in Fragaria spp. and AcRDR1 in pineapple (Ananas comosus) are upregulated by ABA [23,39], whereas FaRDR1d and FaRDR1g are also activated by SA, MeJA, and GA [23]. By contrast, SmRDRs in S. miltiorrhiza are suppressed under MeJA treatment [37]. Intriguingly, previous studies demonstrated that MeJA treatment significantly upregulated the expression of key mogroside biosynthesis enzyme genes (e.g., SgSQS, SgCS, SgCAS, SgCYP23/26/43, and SgGT1/2/4/6/7) in S. grosvenorii, resulting in a 15% increase in mogroside IIE content and a 20% elevation in squalene and cucurbitadienol levels (dry weight, DW) [40], although the regulatory mechanism remains unclear. Based on these findings, we hypothesize that ABA and MeJA might indirectly influence the expression of key mogroside biosynthesis enzyme genes by regulating the amplification and silencing of SgAGO- or SgRDR-dependent siRNA transcripts, which requires further experimental validation.
Analysis of stress-responsive elements further supports the critical role of the RNAi pathway in environmental adaptation. Light-responsive elements are the most abundant (186/244) promoters of SgAGOs, SgDCLs, and SgRDRs, suggesting their involvement in light signal-mediated metabolism or photostress responses, consistent with the predominant distribution of light-responsive elements in C. sinensis [22]. Additionally, the presence of elements related to hypoxia (34/244), low temperature (7/244), and drought (7/244) provides clues for deciphering the molecular mechanisms underlying S. grosvenorii’s adaptation to the unique ecological environment of Guangxi mountainous regions in China. For example, SsAGO10c, SsDCL2, and SsRDR6b in sugarcane (Saccharum spontaneum) were significantly upregulated under PEG-induced dehydration stress [41], while VvAGO1 in straw mushroom (Volvariella volvacea) exhibited elevated expression under cold stress, indicating its role in cold resistance [42]. Notably, the four unique MYBHv1-binding sites in the SgDCL promoter might participate in drought or salt stress response regulation [43,44]. These findings collectively demonstrate that the RNAi core genes in S. grosvenorii coordinately regulate the synthesis of secondary metabolites and adaptation to abiotic stresses by integrating hormonal signaling and stress response networks.

3.3. Functional Divergence of AGO, DCL, and RDR Gene Families in Tissue-Specific Expression and Developmental Regulation in S. grosvenorii

AGOs, DCLs, and RDRs function as regulators of gene silencing and modulate genomic activity in a tissue-specific manner across different developmental stages in plants. AGO proteins serve as the core components of the RISC and act as the primary effectors of the RNAi pathway [19]. This study reveals that all SgAGO genes are ubiquitously expressed in all tissues of S. grosvenorii but show differential expression levels. For instance, AtAGO10 in A. thaliana [45] and CsAGO10c in C. sinensis [35] exhibit high expression in tissues with active meristem development, such as buds and stems, while SgAGO10.1 in S. grosvenorii displays specific high expression in stems, suggesting its potential involvement in stem development regulation. Notably, AtAGO5 in A. thaliana displays significantly higher expression during flower and seed formation compared to other tissues, and CsAGO5a in C. sinensis also shows preferential accumulation in seeds and flowers [35]. Similarly, SgAGO5 in S. grosvenorii exhibits significantly higher expression in pistils, stamens, and 35 DAP fruits than in other tissues, demonstrating that SgAGO5 might play a key role in reproductive organ development. Additionally, AtAGO4/6/9 in A. thaliana mediate RNA-directed DNA methylation (RdDM) by binding with 24 nt siRNAs [46]. Recent studies demonstrated that mCHH methylation levels in S. grosvenorii fruits are significantly elevated during middle-to-late developmental stages compared to early stages. Furthermore, SgAGO4/6 show markedly higher expression in reproductive organs than in vegetative tissues, suggesting that SgAGO4/6 may function similarly to AtAGO4/6 in de novo methylation establishment and may play a critical role in regulating reproductive organ development.
DCL family members exhibit functional redundancy in miRNA/siRNA biogenesis [47]. Although SgDCL1/3 and SgDCL2/4 in S. grosvenorii belong to distinct phylogenetic clades, their expression patterns are similar, showing significant upregulation in female flowers and middle-to-late fruit developmental stages (35 DAP). Unlike A. thaliana [48], Fragaria spp. [23], and C. annuum L. [16], where DCL1/DCL3 predominantly regulate flower development, SgDCL1/4 exhibit the highest expression in female flowers of S. grosvenorii, while SgDCL3 expression is second only to that in 35 DAP fruits, suggesting collective involvement of the SgDCL family in flower and fruit development. Additionally, SgDCL3 may resemble AtDCL3 in A. thaliana by generating siRNAs to guide DNA methylation [46].
The RDR family is critical for dsRNA synthesis during siRNA biogenesis [1]. In S. grosvenorii leaves, SgRDR1.1/1.3 exhibit extremely low expression levels. Previous studies have shown that homologous genes, such as CmeRDR1c1/c2 in melon (Cucumis melo) and CsaRDR1c1/c2 in cucumber (C. sativus), display negligible expression in healthy leaves but are strongly upregulated upon viral infection (e.g., cucumber mosaic virus, CMV), highlighting the essential role of RDR1 in plant antiviral defense [49]. Except for SgRDR1.2, all other SgRDRs are highly expressed in flowers and middle-to-late fruit developmental stages (35 DAP) of S. grosvenorii, implying their cooperative roles in reproductive development. In A. thaliana, AtRDRs regulate female gametophyte development and fertilization processes [50,51]. Notably, the expression pattern of SgRDR2 correlates closely with those of SgAGO4 and SgDCL3, and all three are associated with the RdDM pathway, indicating that SgRDR2 may participate in epigenetic regulation through DNA methylation mechanisms [46].
In summary, the expression of most SgAGOs, SgDCLs, and SgRDRs was upregulated in floral organs (particularly female flowers), with partial genes showing enhanced expression in stems. This pattern was consistent with the expression profiles of CsaAGOs, CsaDCLs, and CsaRDRs in C. sativus [26], indicating functional conservation of RNAi core genes within the family Cucurbitaceae. This study further found that the majority of SgAGOs, SgDCLs, and SgRDRs were upregulated during the 35 DAP fruit developmental stage in S. grosvenorii. Furthermore, as S. grosvenorii is a dioecious plant, differential expression of SgAGOs, SgDCLs, and SgRDRs was observed between female and male flowers; except for SgAGO6, all other genes exhibited higher expression levels in female flowers compared to male flowers. Current research on AGO, DCL, and RDR genes in dioecious plants remains limited. Future studies could elucidate the molecular mechanisms underlying stamen and pistil development by dissecting RNA silencing-mediated gene regulatory networks.

4. Materials and Methods

4.1. Plant Material

S. grosvenorii (cultivar Qingpiguo) plants were cultivated at the Yongfu County cultivation base (Guilin, China; GPS: 24°57′49.32″ N, 110°1′51.01″ E). Roots, stems, leaves, male flowers, female flowers at bloom day, and fruits at 5 DAP, 35 DAP, and 65 DAP were harvested. Three biological replicates per tissue were collected, immediately frozen in liquid nitrogen, and stored at −80 °C.

4.2. Data Collection and Identification of AGO, DCL, and RDR in S. grosvenorii

The protein sequences of A. thaliana AGO, DCL, and RDR were retrieved from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 5 December 2024) as reference sequences. The S. grosvenorii genome data will be published separately. S. grosvenorii homologs were identified using the Basic Local Alignment Search Tool (BLAST) with Hidden Markov Models (HMMs). HMMs generated position-specific scoring matrices to align query sequences against the reference database. Predicted protein sequences were extracted with >40% identity (BLOSUM62 matrix) and E-values < 10 × 10−10. To avoid redundancy, only the longest transcript per locus was retained. Identified genes were named based on phylogenetic relationships with A. thaliana homologs. Physicochemical properties (Mw, pI) were analyzed using ExPASy ProtParam (http://web.expasy.org/protparam, accessed on 7 December 2024). Subcellular localization was predicted via Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 8 December 2024).

4.3. Phylogenetic Tree Construction

Protein sequences of AGO, DCL, and RDR from 14 plant species (Tables S1–S3), including model plants (A. thaliana, Oryza sativa, Zea mays) and Cucurbitaceae (Cucumis sativus, Momordica charantia, Cucumis melo, Citrullus lanatus), were obtained from NCBI and CuGenDBv2 (http://cucurbitgenomics.org/v2/, accessed on 5 January 2025). Multiple sequence alignment was performed using MUSCLE with default settings, specifically a Gap Opening Penalty of −2.90 and a Gap Extension Penalty of 0.00. Subsequently, maximum-likelihood trees were constructed in MEGA 11 (version 11.0.13) utilizing the Poisson correction model accompanied by gamma-distributed rate variation. Bootstrap support values were computed from 1000 replicates initiated with a random seed. The phylogenetic trees were visualized via iTOL (v6.8, https://itol.embl.de, accessed on 10 January 2025), with branch lengths adjusted according to the substitution rate and bootstrap values of ≥50% exhibited at the nodes.

4.4. Conserved Motif and Gene Structure Analysis

Conserved domains in SgAGO, SgDCL, and SgRDR proteins were identified via Pfam (https://pfam.xfam.org/, accessed on 15 January 2025). Conserved motifs in SgAGO, SgDCL, and SgRDR proteins were analyzed using the Multiple Em for Motif Elicitation (MEME) Suite (v5.05) program (http://meme-suite.org, accessed on 15 January 2025) [52]. Promoter regions (2000 bp upstream) of SgAGO, SgDCL, and SgRDR genes were screened for cis-acting elements using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 January 2025). Protein interaction networks were predicted via STRING 12 (https://string-db.org, accessed on 19 January 2025).

4.5. Gene Expression Analysis

Total RNA was isolated using Ve Zol Reagent (R411, Vazyme, Nanjing, China), a TRIzol-like lysis buffer, according to the manufacturer’s protocol. After RNA isolation, residual genomic DNA was digested with DNase I (EN401, Vazyme, Nanjing, China). qRT-PCR primers (Table S4) were designed in Primer Premier 6. Reactions were performed in triplicate on the CFX96™ system (Bio-Rad, Hercules, CA, USA) using SYBR Premix Ex Taq™ (Vazyme). SgUBQ was employed as an internal reference gene due to its stable expression across various tissues and developmental stages of fruits [53]. In qRT-PCR analysis, root tissue was designated as the basal control, and the relative expression levels of other tissues (stems, leaves, flowers, fruits) were normalized to root expression using the 2−ΔΔCt method with three biological replicates. Heatmaps of relative expression levels were generated and visualized using R 4.4.2.

5. Conclusions

This study systematically identified and characterized the RNAi core gene families—SgAGOs, SgDCLs, and SgRDRs—in S. grosvenorii. Phylogenetic analysis demonstrated Cucurbitaceae-specific evolutionary conservation, with these genes clustering closely with homologs from related species. Promoter cis-element analysis revealed the enrichment of hormonal (MeJA, ABA) and stress-responsive (light, hypoxia) elements, pointing to their roles in environmental adaptation. Tissue-specific expression profiling showed predominant upregulation in flowers and mid-stage fruits (35 DAP), while SgAGO10.1 exhibited stem-specific expression and SgRDR2 lacked tissue specificity. Sex-biased expression patterns implicated RNAi in gametophyte development and secondary metabolism. These findings establish RNAi machinery as a critical regulator of mogroside biosynthesis and stress resilience in S. grosvenorii, providing foundational insights for future research.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26115301/s1.

Author Contributions

Z.L. and X.M. designed the study; Y.Z. performed the experiments and data analysis; Y.Z. and C.W. wrote the manuscript; J.S. finalized the figures and tables; C.M. and L.X. helped in S. grosvenorii material collection. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Beijing Municipal Natural Science Foundation (7252244); Guangxi Key R&D Program (GuiKe AB25069119); National Natural Science Foundation of China (82104548; U20A2004); CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-071); Beijing City University 2024 Team Fund (KYTD202401); and Beijing City University “College Students’ Innovation and Entrepreneurship Training Program” (202411418158).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Material.

Conflicts of Interest

The authors declare that there are no conflict of interests.

Abbreviations

The following abbreviations are used in this manuscript:
ABAAbscisic Acid
AGOArgonaute
DAPDays After Pollination
DCLDicer-like
DSRMDouble-stranded RNA-binding domain
dsRNADouble-stranded RNA
GAGibberellin
GRAVYgrand average of hydropathicity
ICLIsocitrate lyase
MeJAMethyl Jasmonate
MEMEMultiple Em for Motif Elicitation
ORFopen reading frame
PPIProtein–protein interaction
qRT-PCRQuantitative real-time PCR
RDRRNA-dependent RNA polymerase
RdDMRNA-directed DNA methylation
RNAiRNA interference
RISCRNA-induced silencing complex
SASalicylic Acid
sRNAssmall RNAs
siRNAssmall interfering RNAs
UBQ Ubiquitin

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Figure 1. Phylogenetic relationships (I), conserved motifs (II), and conserved domains (III) of SgAGO (A), SgDCL (B), and SgRDR (C) families in A. thaliana and S. grosvenorii.
Figure 1. Phylogenetic relationships (I), conserved motifs (II), and conserved domains (III) of SgAGO (A), SgDCL (B), and SgRDR (C) families in A. thaliana and S. grosvenorii.
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Figure 2. Phylogenetic analysis of the SgAGO (A), SgDCL (B), and SgRDR (C) proteins.
Figure 2. Phylogenetic analysis of the SgAGO (A), SgDCL (B), and SgRDR (C) proteins.
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Figure 3. Computational prediction of the protein–protein interaction (PPI) network for SgDCLs, SgAGOs, and SgRDRs. Nodes represent proteins, colored by k-means clustering (Cluster 1: blue; Cluster 2: green; Cluster 3: red). Solid lines denote strong interactions (score > 0.7), while dotted lines indicate weaker associations (score ≤ 0.7). Proteins within the same subfamily are mapped with the highest STRING database identity scores.
Figure 3. Computational prediction of the protein–protein interaction (PPI) network for SgDCLs, SgAGOs, and SgRDRs. Nodes represent proteins, colored by k-means clustering (Cluster 1: blue; Cluster 2: green; Cluster 3: red). Solid lines denote strong interactions (score > 0.7), while dotted lines indicate weaker associations (score ≤ 0.7). Proteins within the same subfamily are mapped with the highest STRING database identity scores.
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Figure 4. Prediction of cis-elements in the 2000 bp upstream regulatory regions of SgAGO (A), SgDCL (B), and SgRDR (C) genes. Different cis-responsive elements are represented by different colored boxes.
Figure 4. Prediction of cis-elements in the 2000 bp upstream regulatory regions of SgAGO (A), SgDCL (B), and SgRDR (C) genes. Different cis-responsive elements are represented by different colored boxes.
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Figure 5. Heatmap illustrating the expression patterns of SgAGO, SgDCL, and SgRDR genes across various organs. Relative expression levels in S. grosvenorii were quantified via qRT-PCR analysis of corresponding organs such as roots, stems, leaves, female flowers, male flowers, and fruits at three ripening stages, with SgUBQ serving as the reference gene. The color scale representing the z-scores is displayed in the right-hand panel, with red indicating values where Z > 1.5 and blue indicating values where Z < −1.5.
Figure 5. Heatmap illustrating the expression patterns of SgAGO, SgDCL, and SgRDR genes across various organs. Relative expression levels in S. grosvenorii were quantified via qRT-PCR analysis of corresponding organs such as roots, stems, leaves, female flowers, male flowers, and fruits at three ripening stages, with SgUBQ serving as the reference gene. The color scale representing the z-scores is displayed in the right-hand panel, with red indicating values where Z > 1.5 and blue indicating values where Z < −1.5.
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Table 1. Basic features of SgAGO, SgDCL, and SgRDR genes.
Table 1. Basic features of SgAGO, SgDCL, and SgRDR genes.
Gene NameGene LocusNumber of Amino AcidsMolecular Weight (kDa)Theoretical pIGrand Average of Hydropathicity (GRAVY)Predicted Subcellular
Localization
SgAGO1Chr09.g173021056117.119.31−0.50Nucleus
SgAGO4Chr06.g106651014113.608.79−0.32Cell membrane, chloroplast, and nucleus
SgAGO5Chr09.g16238967107.549.48−0.41Nucleus
SgAGO7Chr10.g185651018115.589.37−0.40Chloroplast and nucleus
SgAGO10.1Chr08.g15010927105.339.22−0.39Cell membrane and nucleus
SgAGO10.2Chr03.g06089984110.639.33−0.44Nucleus
SgDCL1Chr03.g066421952219.665.87−0.44Nucleus
SgDCL2Chr08.g145521628183.608.55−0.060Nucleus
SgDCL3Chr02.g031041708191.746.39−0.16Nucleus
SgDCL4Chr08.g151251651186.626.30−0.27Nucleus
SgRDR1.1Chr10.g178891112127.797.89−0.32Chloroplast
SgRDR1.2Chr01.g012401123128.688.11−0.38Chloroplast and nucleus
SgRDR1.3Chr10.g178901133129.168.30−0.29Chloroplast
SgRDR2Chr08.g155761121128.816.72−0.29Nucleus
SgRDR5Chr08.g149321026116.357.89−0.30Chloroplast
SgRDR6Chr02.g030341194135.986.97−0.30Chloroplast and nucleus
Table 2. Shared identity between SgAGO, SgDCL, and SgRDR proteins and A. thaliana orthologs based on PPI network analysis.
Table 2. Shared identity between SgAGO, SgDCL, and SgRDR proteins and A. thaliana orthologs based on PPI network analysis.
Query IndexQuery ItemPreferred NameIdentity (%)Bitscore
1SgAGO1AtAGO182.81565.8
2SgAGO4AtAGO472.91357.8
3SgAGO5AtAGO5671164.1
4SgAGO7AtAGO770.21269.6
5SgAGO10.1AtAGO1074.51348.6
6SgAGO10.2AtAGO1082.61661.4
7SgDCL1AtDCL171.72708.3
8SgDCL2AtDCL257.81550
9SgDCL3AtDCL347.81435.6
10SgDCL4AtDCL455.41721.8
11SgRDR1.1AtRDR161.21370.5
12SgRDR1.2AtRDR158.61266.9
13SgRDR1.3AtRDR163.71444.9
14SgRDR2AtRDR261.21403.7
15SgRDR5AtRDR551.3866.3
16SgRDR6AtRDR6671674.1
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Zang, Y.; Wang, C.; Su, J.; Mo, C.; Xie, L.; Luo, Z.; Ma, X. Genome-Wide Identification and Characterization of AGO, DCL, and RDR Gene Families in Siraitia grosvenorii. Int. J. Mol. Sci. 2025, 26, 5301. https://doi.org/10.3390/ijms26115301

AMA Style

Zang Y, Wang C, Su J, Mo C, Xie L, Luo Z, Ma X. Genome-Wide Identification and Characterization of AGO, DCL, and RDR Gene Families in Siraitia grosvenorii. International Journal of Molecular Sciences. 2025; 26(11):5301. https://doi.org/10.3390/ijms26115301

Chicago/Turabian Style

Zang, Yimei, Chongnan Wang, Jiaxian Su, Changming Mo, Lei Xie, Zuliang Luo, and Xiaojun Ma. 2025. "Genome-Wide Identification and Characterization of AGO, DCL, and RDR Gene Families in Siraitia grosvenorii" International Journal of Molecular Sciences 26, no. 11: 5301. https://doi.org/10.3390/ijms26115301

APA Style

Zang, Y., Wang, C., Su, J., Mo, C., Xie, L., Luo, Z., & Ma, X. (2025). Genome-Wide Identification and Characterization of AGO, DCL, and RDR Gene Families in Siraitia grosvenorii. International Journal of Molecular Sciences, 26(11), 5301. https://doi.org/10.3390/ijms26115301

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